Significant Kinetic Solvent Isotope Effects in Folding of the Catalytic RNA from the Hepatitis Delta Virus

Tinsley, Rebecca A.; Harris, D. Ahmasi; Walter, Nils G.

doi:10.1021/ja037870b

Cited by 20 publications

(21 citation statements)

References 9 publications

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“…In contrast, proton inventories are consistent with two protons in flight during the transition state as indicated in the first mechanism, however, the inventory data are sufficiently ambiguous that a one-proton mechanism cannot be discounted (Pinard et al 2001). In addition, the interpretation of proton inventories is complicated by the fact that changes in hydrogen bonding alone can give rise to the underlying kinetic solvent isotope effects (Tinsley et al 2003).…”

Section: Discussionmentioning

confidence: 96%

Molecular dynamics suggest multifunctionality of an adenine imino group in acid-base catalysis of the hairpin ribozyme

Ditzler

Šponer

Walter

2009

RNA

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Despite numerous structural and biochemical investigations, the catalytic mechanism of hairpin ribozyme self-cleavage remains elusive. To gain insight into the coupling of active site dynamics with activity of this small catalytic RNA, we analyzed a total of ;300 ns of molecular dynamics (MD) simulations. Our simulations predict improved global stability for an in vitro selected ''gain of function'' mutation, which is validated by native gel electrophoretic mobility shift assay. We observe that active site nucleobases and water molecules stabilize a geometry favorable to catalysis through a dynamic hydrogen bonding network. Simulations in which A38 is unprotonated show its N1 move into close proximity of the active site 29-OH, indicating that A38 may act as a general base during cleavage, a role that has generally been discounted due to the longer distances observed in crystal structures involving inactivating substrate analogs. By contrast, simulations in which N1 of A38 is protonated place N1 in close proximity to the 59-oxygen leaving group, which supports the proposal that A38 serves as a general acid. In analogy to protein enzymes, we discuss a plausible mechanism in which A38 acts bifunctionally and shuttles a proton directly from the 29-OH to the 59-oxygen. Furthermore, our simulations suggest an important role for protonation of N1 of A38 in promoting a favorable geometry similar to that observed in transition-state analog crystal structures, and support previously proposed roles of A38, G8, and long residency water molecules in transition-state stabilization.

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Section: Discussionmentioning

confidence: 96%

Molecular dynamics suggest multifunctionality of an adenine imino group in acid-base catalysis of the hairpin ribozyme

Ditzler

Šponer

Walter

2009

RNA

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“…This number was very similar to the KSIE observed in MgCl 2 ; this suggests, qualitatively, a similar conclusion for the two cases-that proton transfers were occurring in the rate-determining steps. Here too, however, we cannot formally rule out a role of conformational changes [27] or, in such a complex …”

Section: Kinetic Solvent Isotope Effect Experimentsmentioning

confidence: 91%

The RNA‐Cleaving Bipartite DNAzyme Is a Distinctive Metalloenzyme

et al. 2006

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Much interest has focused on the mechanisms of the five naturally occurring self-cleaving ribozymes, which, in spite of catalyzing the same reaction, adopt divergent strategies. These ribozymes, with the exception of the recently described glmS ribozyme, do not absolutely require divalent metal ions for their catalytic chemistries in vitro. A mechanistic investigation of an in vitro-selected, RNA-cleaving DNA enzyme, the bipartite, which catalyzes the same chemistry as the five natural self-cleaving ribozymes, found a mechanism of significant complexity. The DNAzyme showed a bell-shaped pH profile. A dissection of metal usage indicated the involvement of two catalytically relevant magnesium ions for optimal activity. The DNAzyme was able to utilize manganese(II) as well as magnesium; however, with manganese it appeared to function complexed to either one or two of those cations. Titration with hexaamminecobalt(III) chloride inhibited the activity of the bipartite; this suggests that it is a metalloenzyme that utilizes metal hydroxide as a general base for activation of its nucleophile. Overall, the bipartite DNAzyme appeared to be kinetically distinct not only from the self-cleaving ribozymes but also from other in vitro-selected, RNA-cleaving deoxyribozymes, such as the 8-17, 10-23, and 614.

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“…For the GlcN6P concentration dependence, n = 1 gave the best fit. The pH dependency was fit with equation k obs = k max ð1 + 10 pK a1 ÀpH + 10 pHÀpk a2 Þ as previously described Pereira et al 2002;Tinsley et al 2003).…”

Section: Cleavage Reactionsmentioning

confidence: 99%

Trans-acting glmS catalytic riboswitch: Locked and loaded

Tinsley

Furchak

Walter³

2007

RNA

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A recently discovered class of gene regulatory RNAs, coined riboswitches, are commonly found in noncoding segments of bacterial and some eukaryotic mRNAs. Gene up-or down-regulation is triggered by binding of a small organic metabolite, which typically induces an RNA conformational change. Unique among these noncoding RNAs is the glmS catalytic riboswitch, or ribozyme, found in the 59-untranslated region of the glmS gene in Gram-positive bacteria. It is activated by glucosamine-6-phosphate (GlcN6P), leading to site-specific backbone cleavage of the mRNA and subsequent repression of the glmS gene, responsible for cellular GlcN6P production. Recent biochemical and structural evidence suggests that the GlcN6P ligand acts as a coenzyme and participates in the cleavage reaction without inducing a conformational change. To better understand the role of GlcN6P in solution structural dynamics and function, we have separated the glmS riboswitch core from Bacillus subtilis into a trans-cleaving ribozyme and an externally cleaved substrate. We find that trans cleavage is rapidly activated by nearly 5000-fold to a rate of 4.4 min À1 upon addition of 10 mM GlcN6P, comparable to the cis-acting ribozyme. Fluorescence resonance energy transfer suggests that this ribozyme-substrate complex does not undergo a global conformational change upon ligand binding in solution. In addition, footprinting at nucleotide resolution using terbium(III) and RNase V1 indicates no significant changes in secondary and tertiary structure upon ligand binding. These findings suggest that the glmS ribozyme is fully folded in solution prior to binding its activating ligand, supporting recent observations in the crystalline state.

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Significant Kinetic Solvent Isotope Effects in Folding of the Catalytic RNA from the Hepatitis Delta Virus

Cited by 20 publications

References 9 publications

Molecular dynamics suggest multifunctionality of an adenine imino group in acid-base catalysis of the hairpin ribozyme

Molecular dynamics suggest multifunctionality of an adenine imino group in acid-base catalysis of the hairpin ribozyme

The RNA‐Cleaving Bipartite DNAzyme Is a Distinctive Metalloenzyme

Trans-acting glmS catalytic riboswitch: Locked and loaded

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